EP4308604A1 - Anticorps de liaison à pd-l1 et leurs utilisations - Google Patents

Anticorps de liaison à pd-l1 et leurs utilisations

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Publication number
EP4308604A1
EP4308604A1 EP21931136.2A EP21931136A EP4308604A1 EP 4308604 A1 EP4308604 A1 EP 4308604A1 EP 21931136 A EP21931136 A EP 21931136A EP 4308604 A1 EP4308604 A1 EP 4308604A1
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EP
European Patent Office
Prior art keywords
seq
nos
antibody
antibodies
antigen
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
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EP21931136.2A
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German (de)
English (en)
Inventor
Jiangmei Li
Wenqi Hu
Feng Li
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Beijing Mabworks Biotech Co Ltd
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Beijing Mabworks Biotech Co Ltd
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Publication of EP4308604A1 publication Critical patent/EP4308604A1/fr
Pending legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the disclosure relates to an antibody or an antigen binding portion thereof specifically binding to human PD-L1, the preparation and use thereof, especially its use in treatment of diseases associated with PD-L1 signaling, such as cancers and infectious diseases.
  • T cell-based immunity has evolved to recognize and destroy aberrant cells, including pathogen-infected cells and cancer cells. T cell activities are regulated by a series of co-stimulatory and co-inhibitory receptors as well as their ligands, known as immune checkpoints, which are critical for self-tolerance, preventing the immune system from attacking tissues indiscriminately.
  • PD-1 a type I transmembrane protein that negatively regulates immune responses, is mainly expressed on antigen-experienced memory T cells in peripheral tissues, and less commonly on B cells, activated monocytes, dendritic cells and natural killer cells (Keir ME et al., (2008) Annu Rev Immunol. 26: 677-704; Ishida Y et al., (1992) EMBO J. 11 (11) : 3887-3895) .
  • PD-L1 and PD-L2 are PD-1’s ligands.
  • PD-L1 is a type I transmembrane protein, consisting of two extracellular domains (the IgV-and IgC-like domains) , a transmembrane domain, and an intracellular domain. It is constitutively expressed on antigen-presenting cells, T cells, B cells, monocytes, and epithelial cells, and is upregulated in many types of these cells at the presence of pro-inflammatory cytokines (Keir ME et al., (2008) supra; Chen J et al., (2016) Ann Oncol. 27 (3) : 409-416) . Studies have shown that PD-L1 induces IL-10 release and thus produces inhibitory effects on T cells (Dong H et al., (1999) Nat Med.
  • PD-L2 expression is almost restricted to antigen-presenting cells, and inducible expression can be found on e.g., dendritic cells and macrophages. Accumulating evidence showed PD-1 axis, when engaged with PD-L1, affects cytokine (e.g., IFN- ⁇ , TNF- ⁇ , and IL-2) production to dampen T cell responses against normal cells/tissues (Keir ME et al., (2008) supra; Chen J et al., (2016) supra) .
  • cytokine e.g., IFN- ⁇ , TNF- ⁇ , and IL-2
  • the PD-1-PD-L1 interaction also inhibits secretion of cell survival factors, expression of transcription factors associated with effector cell functions, and lytic activities of activated B cells and NK cells (Terme M et al., (2011) Cancer Res. 71 (16) : 5393-5399; Fanoni D et al., (2011) Immunol Lett. 134 (2) : 157-160) .
  • PD-1 is also highly expressed on regulatory T cells (Treg) , and may promote Treg activation and proliferation, further inhibiting the immune responses (Francisco LM et al., (2009) J Exp Med. 206 (13) : 3015-3029) .
  • PD-1 pathways may be utilized by tumor cells to evade hosts’ immune surveillance.
  • TILs tumor-infiltrating lymphocytes
  • many tumor cells including melanoma, ovarian cancer, lung cancer and renal cancer cells, constitutively express PD-1’s ligands, especially PD-L1 (Dong H et al., (2002) Nat Med. 8 (8) : 793-800; Kim J et al., (2005) Am J Respir Cell Mol Biol. 33 (3) : 280-289; Lee SK et al., (2005) J Dermatol Sci. 40 (2) : 95-103) .
  • PD-L1 is also expressed on myeloid cells, including a subset of macrophages and dendritic cells, in the microenvironment. Therefore, in the microenvironment, PD-L1-PD-1 interaction causes T cell dysfunction and exhaustion, IL-10 release, and reduced cytotoxicity against tumor cells by CD8 + T cells, resulting in tumor cell growth (Zou W, Chen L. (2008) Nat Rev Immunol. 8 (6) : 467-477; Sun Z et al., (2015) Cancer Res. 75 (8) : 1635-1644) .
  • the immune responses may be further depressed by Tregs in the microenvironment (Francisco LM et al., (2009) supra) , and PD-L1-CD80 heterodimers which dampen CD80-CTLA4 interaction (Butte MJ et al., (2007) Immunity. 27 (1) : 111-122; Paterson AM et al., (2011) J Immunol. 187 (3) : 1097-1105) .
  • PD-1/PD-L1 blockade by e.g., antibodies can induce durable tumor remissions in a number of cancers, including solid tumors and hematologic tumors.
  • solid tumors such as melanoma, non-small cell lung cancer (NSCLC) , renal cell carcinoma (RCC) , Hodgkin’s lymphoma, bladder cancer, head and neck carcinoma, neuroendocrine tumor, microsatellite instable-high (MSI-H) and mismatch repair-deficient (dMMR) solid tumors
  • hematologic tumors such as mantle cell lymphoma, diffuse large B-cell lymphoma, and follicular lymphoma (Akinleye A, Rasool Z.
  • Atezolizumab is a humanized IgG1 antibody that is able to block PD-L1-PD-1 and PD-L1-CD80 binding, its Fc region is designed to not induce antibody dependent cell mediated cytotoxicity (ADCC) so as to eliminate damage to PD-L1 + T cells.
  • ADCC antibody dependent cell mediated cytotoxicity
  • Atezolizumab has shown clinical efficacy in solid and non-solid tumors, including NSCLC, melanoma, RCC, colorectal cancer, stomach cancer, head and neck squamous-cell carcinoma, and urothelial carcinoma.
  • the humanized antibody Durvalumab can block PD-L1 binding with PD-1 or CD80, and is designed to avoid ADCC against PD-L1 + T cells. It is efficacious in treating e.g., urothelial carcinoma and NSCLC.
  • Avelumab another humanized IgG1 antibody with Fc region not modified, can block PD-L1-PD-1 and PD-L1- CD80 interaction, induce ADCC against tumor cells, and has shown good clinical outcomes in treatment of Merkel-cell carcinoma, urothelial carcinoma, and three-negative breast cancer.
  • Other anti-PD-L1 antibodies under clinical trials include Envafolimab and BMS-936559.
  • the anti-PD-L1 antibodies may be used in combination with other antibodies, targeted agents, and/or chemotherapeutic agents, to better inhibit tumor growth.
  • the PD-L1/PD-1 blockade also showed good effects in clinical or pre-clinical studies of acute or chronic viral, bacterial, or parasite infections (Jubel JM et al., (2020) Front Immunol. 11: 487) .
  • chronic infections by hepatitis B virus (HBV) hepatitis C virus (HCV)
  • HCV hepatitis C virus
  • HSV human immunodeficiency virus
  • SIV simian immunodeficiency virus
  • administration of anti-PD-L1 antibodies increased IFN- ⁇ , IL-2 and TNF ⁇ release, functionally reversed T cell exhaustion, and ameliorated viremia.
  • more anti-PD-L1 antibodies are also needed for infectious disease treatment.
  • the present disclosure provides an isolated monoclonal antibody, for example, a mouse, human, chimeric or humanized monoclonal antibody, that binds to PD-L1 (e.g., the human PD-L1, and monkey PD-L1) , or an antigen-binding portion thereof. It has comparable or higher binding capability to human and monkey PD-L1 proteins, comparable blocking activity on PD-L1-PD-1 interaction, comparable or better T cell activation capability, and comparable or higher in vivo anti-tumor activity, as compared to prior art antibodies such as Atezolizumab.
  • PD-L1 e.g., the human PD-L1, and monkey PD-L1
  • PD-L1 e.g., the human PD-L1, and monkey PD-L1
  • It has comparable or higher binding capability to human and monkey PD-L1 proteins, comparable blocking activity on PD-L1-PD-1 interaction, comparable or better T cell activation capability, and comparable or higher in vivo anti-
  • the antibody or antigen-binding portion thereof of the disclosure can be used for a variety of applications, including detection of PD-L1 proteins and treatment of PD-L1 associated diseases.
  • the disclosure pertains to an isolated monoclonal antibody (e.g., a humanized antibody) , or an antigen-binding portion thereof, that binds PD-L1, that may comprise i) a heavy chain variable region that may comprise a VH-CDR1 region, a VH-CDR2 region and a VH-CDR3 region, wherein the VH-CDR1 region, the VH-CDR2 region and the VH-CDR3 region may comprise amino acid sequences having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to, or set forth in (1) SEQ ID NOs: 1, 2 and 3, respectively; or (2) SEQ ID NOs: 7, 8 and 9, respectively; and/or ii) a light chain variable region that may comprise a VL-CDR1 region,
  • the isolated monoclonal antibody or antigen-binding portion thereof of the disclosure may comprise a heavy chain variable region and a light chain variable region, wherein the VH-CDR1 region, VH-CDR2 region, VH-CDR3 region, VL-CDR1 region, VL-CDR2 region and VL-CDR3 region may comprise amino acid sequences having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to, or set forth in (1) SEQ ID NOs: 1, 2, 3, 4, 5 and 6, respectively; or (2) SEQ ID NOs: 7, 8, 9, 10, 11 and 12, respectively.
  • the heavy chain variable region of the disclosure may comprise an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to, or set forth in any one of SEQ ID NOs: 13-23.
  • the light chain variable region of the disclosure may comprise an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to, or set forth in any one of SEQ ID NOs: 24-32.
  • the isolated monoclonal antibody or antigen-binding portion thereof of the disclosure may comprise a heavy chain variable region and a light chain variable region which may comprise amino acid sequences having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%95%, 96%, 97%, 98%, or 99%identity to, or set forth in (1) SEQ ID NOs: 13 and 24, respectively; (2) SEQ ID NOs: 14 and 25, respectively; (3) SEQ ID NOs: 14 and 26, respectively; (4) SEQ ID NOs: 14 and 27, respectively; (5) SEQ ID NOs: 15 and 25, respectively; (6) SEQ ID NOs: 15 and 26, respectively; (7) SEQ ID NOs: 15 and 27, respectively; (8) SEQ ID NOs: 16 and 25, respectively; (9) SEQ ID NOs: 16 and 26, respectively; (10) SEQ ID NOs: 16 and 27, respectively; (11) SEQ ID NOs: 17 and 25, respectively; (12) SEQ ID
  • the isolated monoclonal antibody of the disclosure may comprise a heavy chain constant region and/or a light chain constant region.
  • the heavy chain constant region may be an IgG1, IgG2, IgG3 or IgG4 heavy chain constant region, or a functional fragment thereof.
  • the heavy chain constant region may be modified to induce decreased ADCC against PD-L1 + cells, or modified to not induce ADCC against PD-L1 + cells.
  • the heavy chain constant region may be human IgG1 constant region with N297A mutation, having e.g., the amino acid sequence of SEQ ID NO: 33.
  • the light chain constant region may be kappa light chain constant region such as human kappa light chain constant region, having e.g., the amino acid sequence of SEQ ID NO: 34.
  • the N terminus of the heavy chain constant region is linked to the C terminus of the heavy chain variable region, and the N terminus of the light chain constant region is linked to the C terminus of the light chain variable region.
  • the antibody of the disclosure may comprise or consists of two heavy chains and two light chains connected by disulfide bonds, wherein each heavy chain comprises the heavy chain constant region, heavy chain variable region or CDR sequences mentioned above, and each light chain comprises the light chain constant region, light chain variable region or CDR sequences mentioned above.
  • the antibody of the disclosure may be a full-length antibody, for example, of an IgG4, IgG1 or IgG2 isotype.
  • the antibody or antigen binding portion thereof of the present disclosure in other embodiments may be a single chain antibody, or consists of antibody fragments, such as Fab or F (ab′) 2 fragments.
  • the exemplary antibody or antigen binding portion thereof of the disclosure is antagonistic, which binds to human/monkey PD-L1, blocks PD-L1-PD-1 interaction, induces T cell activation, and/or has in vivo anti-tumor effects.
  • the disclosure also provides an immuneconjugate comprising the antibody or the antigen binding portion thereof, linked to a therapeutic agent such as a cytotoxin or an anti-cancer agent.
  • the disclosure also provides a bispecific molecule comprising the antibody or the antigen-binding portion thereof of the disclosure, linked to a second functional moiety (e.g., a second antibody) having a different binding specificity than the antibody or the antigen-binding portion thereof of the disclosure.
  • the antibody or the antigen-binding portion thereof of the present disclosure can be made into part of a chimeric antigen receptor (CAR) or a T cell receptor (TCR) .
  • the disclosure further provides an immune cell with the CAR or TCR of the disclosure, such as a T cell and a NK cell.
  • the antibody or antigen binding portion thereof of the disclosure can also be encoded by or used in conjunction with an oncolytic virus.
  • the disclosure further provides a nucleic acid molecule encoding the antibody or antigen-binding portion thereof of the disclosure, as well as an expression vector comprising such a nucleic acid molecule and a host cell comprising such an expression vector.
  • a method for preparing the anti-PD-L1 antibody or antigen binding portion thereof using the host cell of the disclosure comprising steps of (i) expressing the antibody or antigen binding portion thereof in the host cell, and (ii) isolating the antibody or antigen binding portion thereof from the host cell or its cell culture.
  • the disclosure provides a composition comprising the antibody or antigen binding portion thereof, the immuneconjugate, the bispecific molecule, the immune cell, the oncolytic virus, the nucleic acid molecule, the expression vector, or the host cell of the disclosure, and a pharmaceutically acceptable carrier.
  • the disclosure provides a method for enhancing an immune response in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the composition of the disclosure.
  • the method comprises inducing T cell activation.
  • the disclosure provides a method for treating or alleviating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the composition of the disclosure.
  • the cancer may be a solid cancer or a hematological cancer, including, but not limited to, melanoma, non-small cell lung cancer, renal cell carcinoma, Hodgkin lymphoma, bladder cancer, head and neck cancer, neuroendocrine tumor, mantle cell lymphoma, diffuse large B-cell lymphoma, and follicular lymphoma.
  • At least one additional anti-cancer antibody may be administered with the composition of the disclosure, such as an anti-PD-1 antibody, an anti-STAT3 antibody, an anti-ROR1 antibody, an anti-TIM-3 antibody, and/or an anti-CTLA-4 antibody.
  • the composition of the disclosure may be administered with a cytokine (e.g., IL-2 and/or IL-21) , or a costimulatory antibody (e.g., an anti-CD137 and/or anti-GITR antibody) .
  • the composition of the disclosure may be administered with a chemotherapeutic agent, which may be a cytotoxic agent.
  • the disclosure provides a method for treating or alleviating an infectious disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the antibody or antigen binding portion thereof.
  • the infectious disease may be a chronic viral, bacterial, fungal or mycoplasma infection, such as a chronic hepatitis B virus (HBV) , hepatitis C virus (HCV) , human immunodeficiency virus (HIV) , or simian immunodeficiency virus (SIV) infection.
  • at least one additional anti-infective agent may be administered with the antibody or antigen binding portion thereof of the disclosure, such as an anti-viral agent, an anti-bacterial agent, an anti-fungal agent, or an anti-mycoplasma agent.
  • FIG. 1 shows the treatment of mouse anti-PD-L1 antibodies at 100 ⁇ g/ml (A) or at 0.1-10 ⁇ g/ml (B) increased IFN- ⁇ secretion by T cells.
  • FIG. 2 shows the binding capabilities of chimeric anti-PD-L1 antibodies to HEK293A/human PD-L1 cells (A) , HEK293A/monkey PD-L1 cells (B) , and HEK293A/mouse PD-L1 cells (C) .
  • FIG. 3 shows the capabilities of chimeric anti-PD-L1 antibodies to block PD-1-PD-L1 interaction.
  • FIG. 4 shows chimeric anti-PD-L1 antibodies induced IFN- ⁇ secretion by T cells in a dose dependent manner.
  • FIG. 5 shows the binding capabilities of humanized 3C2 antibodies to HEK293A/human PD-L1 cells (A) , HEK293A/monkey PD-L1 cells (B) , and HEK293A/mouse PD-L1 cells (C) .
  • FIG. 6 shows the binding capabilities of humanized 56E5 antibodies to HEK293A/human PD-L1 cells (A) , HEK293A/monkey PD-L1 cells (B) , and HEK293A/mouse PD-L1 cells (C) .
  • FIG. 7 shows humanized 56E5 and 3C2 antibodies induced IFN- ⁇ secretion by T cells in a dose dependent manner.
  • FIG. 8 shows the SPR curves of the anti-PDL1 antibody 56E5VH5VL4 in competition with 3C2VH4VL4 (A) , 56E5VH5VL4 (B) , Atezolizumab (C) , Avelumab (D) and Duralumab (E) over PD-L1 binding.
  • FIG. 9 shows the SPR curves of the anti-PDL1 antibody 3C2VH4VL4 in competition with 3C2VH4VL4 (A) , 56E5VH5VL4 (B) , Atezolizumab (C) , Avelumab (D) and Duralumab (E) over PD-L1 binding.
  • FIG. 10 shows the average tumor volumes (A) and average tumor weights (B) of transgenic mice with human PD-L1 in groups respectively treated with 56E5VH5VL4, 3C2VH6VL5 and Avelumab.
  • PD-L1 refers to programmed death-ligand 1.
  • the term “PD-L1” comprises variants, isoforms, homologs, orthologs and paralogs.
  • an antibody specific for a human PD-L1 protein may, in certain cases, cross-react with a PD-L1 protein from a species other than human, such as monkey.
  • an antibody specific for a human PD-L1 protein may be completely specific for the human PD-L1 protein and exhibit no cross-reactivity to other species or of other types, or may cross-react with PD-L1 from certain other species but not all other species.
  • human PD-L1 refers to a PD-L1 protein having an amino acid sequence from a human, such as the amino acid sequence having a GenBank accession no. AAI13735.1 (Strausberg R.L. et al., (2002) Proc. Natl. Acad. Sci. U.S.A. 99 (26) : 16899-16903) or set forth in SEQ ID NO: 35.
  • the term “monkey PD-L1” refers to a PD-L1 protein having an amino acid sequence from a monkey, such as the amino acid sequence having a NCBI accession no. XP_005581836.1 or set forth in SEQ ID NO: 36.
  • mouse PD-L1 refers to a PD-L1 protein having an amino acid sequence from a mouse, such as the amino acid sequence having a GenBank accession no. AAH66841.1 (Strausberg R.L. et al., (2002) supra) or set forth in SEQ ID NO: 37.
  • antibody as referred to herein includes whole antibodies of e.g., IgG, IgA, IgD, IgE and IgM, and any antigen binding fragment (i.e., “antigen-binding portion” ) or single chains thereof.
  • Whole antibodies are glycoproteins comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds.
  • Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as V H ) and a heavy chain constant region.
  • the heavy chain constant region is comprised of three domains, C H1 , C H2 and C H3 .
  • Each light chain is comprised of a light chain variable region (abbreviated herein as V L ) and a light chain constant region.
  • the light chain constant region is comprised of one domain, C L .
  • the V H and V L regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR) , interspersed with regions that are more conserved, termed framework regions (FR) .
  • CDR complementarity determining regions
  • FR framework regions
  • Each V H and V L is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first
  • antibody portion refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., a PD-L1 protein) . It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.
  • binding fragments encompassed within the term “antigen-binding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the V L V H , C L and C H1 domains; (ii) a F (ab′) 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the V H and C H1 domains; (iv) a Fv fragment consisting of the V L and V H domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341: 544-546) , which consists of a V H domain; (vi) an isolated complementarity determining region (CDR) ; and (viii) a nanobody, a heavy chain variable region containing a single variable domain and two constant domains.
  • a Fab fragment a monovalent fragment consisting of the V L V H
  • the two domains of the Fv fragment, V L and V H are coded by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the V L and V H regions pair to form monovalent molecules (known as single chain Fv (scFv) ; see e.g., Bird et al., (1988) Science 242: 423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883) .
  • Such single chain antibodies are also intended to be encompassed within the term “antigen-binding portion” of an antibody.
  • These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.
  • an “isolated antibody” is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds a PD-L1 protein is substantially free of antibodies that specifically bind antigens other than PD-L1 proteins) .
  • An isolated antibody that specifically binds a human PD-L1 protein may, however, have cross-reactivity to other antigens, such as PD-L1 proteins from other species.
  • an isolated antibody can be substantially free of other cellular material and/or chemicals.
  • the term “monoclonal antibody” as used herein refers to a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or post-translation modifications (e.g., isomerization, amidation) that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In contrast to polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes) , the monoclonal antibodies are directed against a single determinant on the antigen.
  • mouse antibody is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from mouse germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from mouse germline immunoglobulin sequences.
  • the mouse antibodies of the disclosure can include amino acid residues not encoded by mouse germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo) .
  • the term “mouse antibody” is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species have been grafted onto mouse framework sequences.
  • chimeric antibody refers to an antibody made by combining genetic material from a nonhuman source with genetic material from a human being. Or more generally, a chimeric antibody is an antibody having genetic material from a certain species with genetic material from another species.
  • humanized antibody refers to an antibody from non-human species whose protein sequences have been modified to increase similarity to antibody variants produced naturally in humans.
  • an antibody recognizing an antigen and “an antibody specific for an antigen” are used interchangeably herein with the term “an antibody which binds specifically to an antigen. ”
  • an antibody that “specifically binds to human PD-L1” is intended to refer to an antibody that binds to human PD-L1 protein (and possibly a PD-L1 protein from one or more non-human species) but does not substantially bind to non-PD-L1 proteins.
  • the antibody binds to human PD-L1 protein with “high affinity” , namely with a K D of 5.0 x10 -8 M or less, more preferably 1.0 x 10 -9 M or less.
  • does not substantially bind to a protein or cells, as used herein, means does not bind or does not bind with a high affinity to the protein or cells, i.e. binds to the protein or cells with a K D of 1.0 x 10 -6 M or more, more preferably 1.0 x 10 -5 M or more, more preferably 1.0 x 10 -4 M or more, more preferably 1.0 x 10 -3 M or more, even more preferably 1.0 x 10 -2 M or more.
  • high affinity for an IgG antibody refers to an antibody having a K D of 1.0 x 10 -6 M or less, more preferably 5.0 x 10 -8 M or less, even more preferably 1.0 x 10 -8 M or less, even more preferably 5.0 x 10 -9 M or less and even more preferably 1.0 x 10 -9 M or less for a target antigen.
  • “high affinity” binding can vary for other antibody isotypes.
  • “high affinity” binding for an IgM isotype refers to an antibody having a K D of 10 -6 M or less, more preferably 10 -7 M or less, even more preferably 10 -8 M or less.
  • K assoc or “K a ”
  • K dis or “K d ”
  • K D is intended to refer to the dissociation rate of a particular antibody-antigen interaction
  • K D is intended to refer to the dissociation constant, which is obtained from the ratio of K d to K a (i.e., K d /K a ) and is expressed as a molar concentration (M) .
  • K D values for antibodies can be determined using methods well established in the art. A preferred method for determining the K D of an antibody is by using surface plasmon resonance, preferably using a biosensor system such as a Biacore TM system.
  • EC 50 also known as half maximal effective concentration, refers to the concentration of an antibody which induces a response halfway between the baseline and maximum after a specified exposure time.
  • antibody dependent cellular cytotoxicity refers to a mechanism of cell-mediated immune defense whereby an effector cell of the immune system actively lyses a target cell bound by antibodies.
  • subject includes any human or nonhuman animal.
  • nonhuman animal includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles, although mammals are preferred, such as non-human primates, sheep, dogs, cats, cows and horses.
  • antagonistic PD-L1 antibody refers to an anti-PD-L1 antibody that binds to PD-L1 and blocks PD-L1 signaling induced by PD-L1’s interaction with its ligands such as PD-1.
  • the antagonistic anti-PD-L1 antibody may promote T cell activation and cytokine release, enhance immunity, and thus can be used to treat e.g., cancers and chronic infections.
  • therapeutically effective amount means an amount of the antibody or antigen binding portion thereof of the present disclosure sufficient to prevent or ameliorate the symptoms associated with a disease or condition (such as a cancer) and/or lessen the severity of the disease or condition.
  • a therapeutically effective amount is understood to be in context of the condition being treated, where the actual effective amount is readily discerned by those of skill in the art.
  • the exemplary antibodies, or antigen binding portions thereof, of the disclosure specifically bind to human and monkey PD-L1 proteins with high binding capabilities that are similar to or higher than those of prior art anti-PD-L1 antibodies such as Atezolizumab.
  • the antibodies or antigen binding portions thereof of the disclosure may block PD-L1-PD-1 binding or interaction, and the blocking activities are comparable to those of prior art anti-PD-L1 antibodies such as Atezolizumab.
  • the antibodies or antigen binding portions thereof of the disclosure have comparable, if not higher, T cell activating capabilities and in vivo anti-tumor activities, compared to prior art anti-PD-L1 antibodies such as Atezolizumab.
  • antibodies or antigen binding portions thereof of the disclosure are monoclonal. Additionally, the antibodies or antigen binding portions thereof can be, for example, mouse, chimeric or humanized.
  • the exemplary antibodies or antigen binding portions thereof of the disclosure are structurally and chemically characterized below.
  • the sequence ID numbers of their heavy chain and light chain variable regions and CDRs are summarized in Table 1, some antibodies or antigen binding portions thereof share the same heavy/light chain variable regions.
  • the heavy chain variable region CDRs and the light chain variable region CDRs in Table 1 have been defined by the Kabat numbering system. However, as is well known in the art, CDR regions can also be determined by other systems such as Chothia, IMGT, AbM, or Contact numbering system/method, based on heavy chain/light chain variable region sequences.
  • the antibodies of the disclosure may each comprise a heavy chain constant region, such as IgG1 constant region, e.g., human IgG1 constant region having the amino acid sequence of SEQ ID NO: 33, or a functional fragment thereof.
  • the antibodies of the disclosure may each comprise a light chain constant region, such as kappa light chain constant region, e.g., human kappa constant region having the amino acid sequence of SEQ ID NO: 34, or a functional fragment thereof.
  • V H and V L sequences (or CDR sequences) of other anti-PD-L1 antibodies which bind to human PD-L1 can be “mixed and matched” with the V H and V L sequences (or CDR sequences) of the anti-PD-L1 antibody of the present disclosure.
  • V H and V L chains or the CDRs within such chains
  • a V H sequence from a particular V H /V L pairing is replaced with a structurally similar V H sequence.
  • a V L sequence from a particular V H /V L pairing is replaced with a structurally similar V L sequence.
  • an antibody of the disclosure, or an antigen binding portion thereof comprises:
  • an antibody of the disclosure, or an antigen binding portion thereof comprises:
  • the antibody, or antigen binding portion thereof includes the heavy chain variable CDR2 region of anti-PD-L1 antibody combined with CDRs of other antibodies which bind human PD-L1, e.g., CDR1 and/or CDR3 from the heavy chain variable region, and/or CDR1, CDR2, and/or CDR3 from the light chain variable region of a different anti-PD-L1 antibody.
  • the CDR3 domain independently from the CDR1 and/or CDR2 domain (s) , alone can determine the binding specificity of an antibody for a cognate antigen and that multiple antibodies can predictably be generated having the same binding specificity based on a common CDR3 sequence. See, e.g., Klimka et al., , British J. of Cancer 83 (2) : 252-260 (2000) ; Beiboer et al., , J. Mol. Biol. 296: 833-849 (2000) ; Rader et al., , Proc. Natl. Acad. Sci. U.S.A.
  • the antibody or antigen-binding portion thereof of the disclosure comprise the CDR2 of the heavy chain variable region of the anti-PD-L1 antibody and at least the CDR3 of the heavy and/or light chain variable region of the anti-PD-L1 antibody, or the CDR3 of the heavy and/or light chain variable region of another anti-PD-L1 antibody, wherein the antibody or antigen binding portion thereof is capable of specifically binding to human PD-L1.
  • These antibodies or antigen binding portions thereof preferably (a) compete for binding with PD-L1; (b) retain the functional characteristics; (c) bind to the same epitope; and/or (d) have a similar binding affinity as the anti-PD-L1 antibody of the present disclosure.
  • the antibodies or antigen binding portions thereof further may comprise the CDR2 of the light chain variable region of the anti-PD-L1 antibody, or the CDR2 of the light chain variable region of another anti-PD-L1 antibody, wherein the antibody or antigen binding portion thereof is capable of specifically binding to human PD-L1.
  • the antibodies or antigen binding portions thereof of the disclosure may include the CDR1 of the heavy and/or light chain variable region of the anti-PD-L1 antibody, or the CDR1 of the heavy and/or light chain variable region of another anti-PD-L1 antibody, wherein the antibody or antigen binding portion thereof is capable of specifically binding to human PD-L1.
  • an antibody or antigen binding portion thereof of the disclosure comprises a heavy and/or light chain variable region sequences of CDR1, CDR2 and CDR3 sequences which differ from those of the anti-PD-L1 antibodies or antigen binding portions thereof of the present disclosure by one or more conservative modifications. It is understood in the art that certain conservative sequence modification can be made which do not remove antigen binding. See, e.g., Brummell et al., (1993) Biochem 32: 1180-8; de Wildt et al., (1997) Prot. Eng. 10: 835-41; Komissarov et al., (1997) J. Biol. Chem. 272: 26864-26870; Hall et al., (1992) J. Immunol.
  • the antibody or antigen binding portion thereof comprises a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences and/or a light chain variable region comprising CDR1, CDR2, and CDR3 sequences, wherein:
  • the heavy chain variable region CDR1 sequence comprises a sequence listed in Table 1 above, and/or conservative modifications thereof;
  • the heavy chain variable region CDR2 sequence comprises a sequence listed in Table 1 above, and/or conservative modifications thereof; and/or
  • the heavy chain variable region CDR3 sequence comprises a sequence listed in Table 1 above, and conservative modifications thereof;
  • the light chain variable region CDR1, and/or CDR2, and/or CDR3 sequences comprise the sequence (s) listed in Table 1 above; and/or conservative modifications thereof;
  • the antibody specifically binds human PD-L1.
  • the antibody or antigen binding portion thereof of the present disclosure possesses one or more of the following functional properties described above, such as high affinity binding to human PD-L1, and reduced or eliminated ability to induce antibody dependent cell mediated cytotoxicity (ADCC) against PD-L1 positive cells.
  • ADCC antibody dependent cell mediated cytotoxicity
  • the antibody or antigen binding portion thereof, of the disclosure can be, for example, mouse, chimeric, human, or humanized.
  • conservative sequence modifications is intended to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody of the disclosure by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • one or more amino acid residues within the CDR regions of an antibody of the disclosure can be replaced with other amino acid residues from the same side chain family and the altered antibody can be tested for retained function (i.e., the functions set forth above) using the functional assays described herein.
  • the antibody of the disclosure can be prepared using an antibody having one or more of the V H /V L sequences of the anti-PD-L1 antibody of the present disclosure as starting material to engineer a modified antibody.
  • An antibody can be engineered by modifying one or more residues within one or both variable regions (i.e., V H and/or V L ) , for example within one or more CDR regions and/or within one or more framework regions. Additionally or alternatively, an antibody can be engineered by modifying residues within the constant region (s) , for example to alter the effector function (s) of the antibody.
  • CDR grafting can be used to engineer variable regions of antibodies.
  • Antibodies interact with target antigens predominantly through amino acid residues that are located in the six heavy and light chain complementarity determining regions (CDRs) . For this reason, the amino acid sequences within CDRs are more diverse between individual antibodies than sequences outside of CDRs.
  • CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of specific naturally occurring antibodies by constructing expression vectors that include CDR sequences from the specific naturally occurring antibody grafted onto framework sequences from a different antibody with different properties (see, e.g., Riechmann et al., (1998) Nature332: 323-327; Jones et al., (1986) Nature 321: 522-525; Queen et al., (1989) Proc. Natl. Acad. See also U.S.A. 86: 10029-10033; U.S. Pat. Nos. 5,225,539; 5,530,101; 5,585,089; 5,693,762 and 6,180,370) .
  • another embodiment of the disclosure pertains to an isolated monoclonal antibody, or antigen binding portion thereof, comprising a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences comprising the sequences of the present disclosure, as described above, and/or a light chain variable region comprising CDR1, CDR2, and CDR3 sequences comprising the sequences of the present disclosure, as described above. While these antibodies contain the V H and V L CDR sequences of the monoclonal antibody of the present disclosure, they can contain different framework sequences.
  • Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences.
  • germline DNA sequences for human heavy and light chain variable region genes can be found in the “VBase” human germline sequence database (available on the Internet at www. mrc-cpe. cam. ac. uk/vbase) , as well as in Kabat et al., (1991) , cited supra; Tomlinson et al., (1992) J. Mol. Biol. 227: 776-798; and Cox et al., (1994) Eur. J. Immunol. 24: 827-836; the contents of each of which are expressly incorporated herein by reference.
  • the germline DNA sequences for human heavy and light chain variable region genes can be found in the Genbank database.
  • the following heavy chain germline sequences found in the HCo7 HuMAb mouse are available in the accompanying Genbank Accession Nos.: 1-69 (NG--0010109, NT--024637 & BC070333) , 3-33 (NG--0010109 & NT--024637) and 3-7 (NG--0010109 & NT--024637) .
  • the following heavy chain germline sequences found in the HCo12 HuMAb mouse are available in the accompanying Genbank Accession Nos.: 1-69 (NG--0010109, NT--024637 & BC070333) , 5-51 (NG--0010109 & NT--024637) , 4-34 (NG--0010109 & NT--024637) , 3-30.3 (CAJ556644) & 3-23 (AJ406678) .
  • Antibody protein sequences are compared against a compiled protein sequence database using one of the sequence similarity searching methods called the Gapped BLAST (Altschul et al., (1997) , supra) , which is well known to those skilled in the art.
  • V H CDR1, CDR2, and CDR3 sequences can be grafted onto framework regions that have the identical sequence as that found in the germline immunoglobulin gene from which the framework sequence derives, or the CDR sequences can be grafted onto framework regions that contain one or more mutations as compared to the germline sequences. For example, it has been found that in certain instances it is beneficial to mutate residues within the framework regions to maintain or enhance the antigen binding ability of the antibody (see e.g., U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370) .
  • variable region modification is to mutate amino acid residues within the V H and/or V L CDR1, CDR2 and/or CDR3 regions to thereby improve one or more binding properties (e.g., affinity) of the antibody of interest.
  • Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce the mutation (s) and the effect on antibody binding, or other functional property of interest, can be evaluated in in vitro or in vivo assays as known in the art.
  • conservative modifications are introduced.
  • the mutations can be amino acid substitutions, additions or deletions, but are preferably substitutions.
  • typically no more than one, two, three, four or five residues within a CDR region are altered.
  • the disclosure provides isolated anti-PD-L1 monoclonal antibodies, or antigen binding portions thereof, comprising a heavy chain variable region comprising: (a) a V H CDR1 region comprising the sequence of the present disclosure, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions; (b) a V H CDR2 region comprising the sequence of the present disclosure, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions; (c) a V H CDR3 region comprising the sequence of the present disclosure, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions; (d) a V L CDR1 region comprising the sequence of the present disclosure, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions; (e) a V L CDR2 region comprising the sequence of the present disclosure, or an amino acid sequence having one, two,
  • Engineered antibodies of the disclosure include those in which modifications have been made to framework residues within V H and/or V L , e.g. to improve the properties of the antibody. Typically, such framework modifications are made to decrease the immunogenicity of the antibody. For example, one approach is to “backmutate” one or more framework residues to the corresponding germline sequence. More specifically, an antibody that has undergone somatic mutation can contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequences to the germline sequences from which the antibody is derived.
  • Another type of framework modification involves mutating one or more residues within the framework region, or even within one or more CDR regions, to remove T cell epitopes to thereby reduce the potential immunogenicity of the antibody. This approach is also referred to as “deimmunization” and is described in further detail in U.S. Patent Publication No. 20030153043.
  • antibodies of the disclosure can be engineered to include modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity.
  • modifications within the Fc region typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity.
  • an antibody of the disclosure can be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or be modified to alter its glycosylation, again to alter one or more functional properties of the antibody.
  • the hinge region of C H1 is modified in such that the number of cysteine residues in the hinge region is altered, e.g., increased or decreased. This approach is described further in U.S. Pat. No. 5,677,425.
  • the number of cysteine residues in the hinge region of C H1 is altered to, for example, facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody.
  • the Fc hinge region of an antibody is mutated to decrease the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the C H2 -C H3 domain interface region of the Fc-hinge fragment such that the antibody has impaired Staphylococcyl protein A (SpA) binding relative to native Fc-hinge domain SpA binding.
  • SpA Staphylococcyl protein A
  • the glycosylation of an antibody is modified.
  • a glycosylated antibody can be made (i.e., the antibody lacks glycosylation) .
  • Glycosylation can be altered to, for example, increase the affinity of the antibody for antigen.
  • Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence.
  • one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site.
  • Such aglycosylation may increase the affinity of the antibody for antigen. See, e.g., U.S. Pat. Nos. 5,714,350 and 6,350,861.
  • an antibody can be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or an antibody having increased bisecting GlcNac structures.
  • altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies.
  • carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies of the disclosure to thereby produce an antibody with altered glycosylation.
  • the cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene, FUT8 ( ⁇ (1, 6) -fucosyltransferase) , such that antibodies expressed in the Ms704, Ms705, and Ms709 cell lines lack fucose on their carbohydrates.
  • the Ms704, Ms705, and Ms709 FUT8-/-cell lines were created by the targeted disruption of the FUT8 gene in CHO/DG44 cells using two replacement vectors (see U.S. Patent Publication No. 20040110704 and Yamane-Ohnuki et al., (2004) Biotechnol Bioeng 87: 614-22) .
  • EP 1,176,195 describes a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyl transferase, such that antibodies expressed in such a cell line exhibit hypofucosylation by reducing or eliminating the ⁇ -1, 6 bond-related enzyme.
  • EP 1, 176, 195 also describes cell lines which have a low enzyme activity for adding fucose to the N-acetylglucosamine that binds to the Fc region of the antibody or does not have the enzyme activity, for example the rat myeloma cell line YB2/0 (ATCC CRL 1662) .
  • PCT Publication WO 03/035835 describes a variant CHO cell line, Lec13 cells, with reduced ability to attach fucose to Asn (297) -linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in that host cell (see also Shields et al., (2002) J. Biol. Chem. 277: 26733-26740) .
  • Antibodies with a modified glycosylation profile can also be produced in chicken eggs, as described in PCT Publication WO 06/089231.
  • antibodies with a modified glycosylation profile can be produced in plant cells, such as Lemna.
  • PCT Publication WO 99/54342 describes cell lines engineered to express glycoprotein-modifying glycosyl transferases (e.g., ⁇ (1, 4) -N-acetylglucosaminyltransferase III (GnTIII) ) such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures which results in increased ADCC activity of the antibodies (see also Umana et al., (1999) Nat. Biotech. 17: 176-180) .
  • glycoprotein-modifying glycosyl transferases e.g., ⁇ (1, 4) -N-acetylglucosaminyltransferase III (GnTIII)
  • the fucose residues of the antibody can be cleaved off using a fucosidase enzyme; e.g., the fucosidase ⁇ -L-fucosidase removes fucosyl residues from antibodies (Tarentino et al., (1975) Biochem. 14: 5516-23) .
  • a fucosidase enzyme e.g., the fucosidase ⁇ -L-fucosidase removes fucosyl residues from antibodies (Tarentino et al., (1975) Biochem. 14: 5516-23) .
  • An antibody can be pegylated to, for example, increase the biological (e.g., serum) half-life of the antibody.
  • the antibody, or fragment thereof typically is reacted with polyethylene glycol (PEG) , such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody or antibody fragment.
  • PEG polyethylene glycol
  • the pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer) .
  • polyethylene glycol is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (C 1 -C 10 ) alkoxy-or aryloxy-polyethylene glycol or polyethylene glycol-maleimide.
  • the antibody to be pegylated is an aglycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to the antibodies of the disclosure. See, e.g., EPO 154 316 and EP 0 401 384.
  • Antibodies of the disclosure can be characterized by their various physical properties, to detect and/or differentiate different classes thereof.
  • antibodies can contain one or more glycosylation sites in either the light or heavy chain variable region. Such glycosylation sites may result in increased immunogenicity of the antibody or an alteration of the pK of the antibody due to altered antigen binding (Marshall et al (1972) Annu Rev Biochem 41: 673-702; Gala and Morrison (2004) J Immunol 172: 5489-94; Wallick et al (1988) J Exp Med 168: 1099-109; Spiro (2002) Glycobiology 12: 43R-56R; Parekh et al (1985) Nature 316: 452-7; Mimura et al., (2000) Mol Immunol 37: 697-706) .
  • Glycosylation has been known to occur at motifs containing an N-X-S/T sequence.
  • an anti-PD-L1 antibody that does not contain variable region glycosylation. This can be achieved either by selecting antibodies that do not contain the glycosylation motif in the variable region or by mutating residues within the glycosylation region.
  • the antibodies do not contain asparagine isomerism sites.
  • the deamidation of asparagine may occur on N-G or D-G sequences and result in the creation of an isoaspartic acid residue that introduces a kink into the polypeptide chain and decreases its stability (isoaspartic acid effect) .
  • Each antibody will have a unique isoelectric point (pI) , which generally falls in the pH range between 6 and 9.5.
  • the pI for an IgG1 antibody typically falls within the pH range of 7-9.5 and the pI for an IgG4 antibody typically falls within the pH range of 6-8.
  • pI isoelectric point
  • the disclosure provides nucleic acid molecules that encode heavy and/or light chain variable regions, or CDRs, of the antibodies of the disclosure.
  • the nucleic acids can be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form.
  • a nucleic acid is “isolated” or “rendered substantially pure” when purified away from other cellular components or other contaminants, e.g., other cellular nucleic acids or proteins, by standard techniques.
  • a nucleic acid of the disclosure can be, e.g., DNA or RNA and may or may not contain intronic sequences.
  • the nucleic acid is a cDNA molecule.
  • Nucleic acids of the disclosure can be obtained using standard molecular biology techniques.
  • cDNAs encoding the light and heavy chains of the antibody made by the hybridoma can be obtained by standard PCR amplification or cDNA cloning techniques.
  • antibodies obtained from an immunoglobulin gene library e.g., using phage display techniques
  • a nucleic acid encoding such antibodies can be recovered from the gene library.
  • Preferred nucleic acids molecules of the disclosure include those encoding the V H and V L sequences of the PD-L1 monoclonal antibody or the CDRs.
  • V H and V L segments Once DNA fragments encoding V H and V L segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example to convert the variable region genes to full-length antibody chain genes, to Fab fragment genes or to a scFv gene.
  • a V L -or V H -encoding DNA fragment is operatively linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker.
  • the term “operatively linked” is intended to mean that the two DNA fragments are joined such that the amino acid sequences encoded by the two DNA fragments remain in-frame.
  • the isolated DNA encoding the V H region can be converted to a full-length heavy chain gene by operatively linking the V H -encoding DNA to another DNA molecule encoding heavy chain constant regions (C H1 , C H2 and C H3 ) .
  • the sequences of human heavy chain constant region genes are known in the art and DNA fragments encompassing these regions can be obtained by standard PCR amplification.
  • the heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most preferably is an IgG1 or IgG4 constant region.
  • the V H -encoding DNA can be operatively linked to another DNA molecule encoding only the heavy chain C H1 constant region.
  • the isolated DNA encoding the V L region can be converted to a full-length light chain gene (as well as a Fab light chain gene) by operatively linking the V L -encoding DNA to another DNA molecule encoding the light chain constant region, C L .
  • the sequences of human light chain constant region genes are known in the art and DNA fragments encompassing these regions can be obtained by standard PCR amplification.
  • the light chain constant region can be a kappa or lambda constant region.
  • the V H -and V L -encoding DNA fragments are operatively linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly4-Ser) 3, such that the V H and V L sequences can be expressed as a contiguous single-chain protein, with the V L and V H regions joined by the flexible linker (see e.g., Bird et al., (1988) Science 242: 423-426; Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883; McCafferty et al., , (1990) Nature 348: 552-554) .
  • a flexible linker e.g., encoding the amino acid sequence (Gly4-Ser) 3, such that the V H and V L sequences can be expressed as a contiguous single-chain protein, with the V L and V H regions joined by the flexible linker (see e.g., Bird
  • Monoclonal antibodies (mAbs) of the present disclosure can be produced using the well-known somatic cell hybridization (hybridoma) technique of Kohler and Milstein (1975) Nature256: 495.
  • Other embodiments for producing monoclonal antibodies include viral or oncogenic transformation of B lymphocytes and phage display techniques.
  • Chimeric or humanized antibodies are also well known in the art. See e.g., U.S. Pat. Nos. 4,816,567; 5,225,539; 5,530,101; 5,585,089; 5,693,762 and 6,180,370, the contents of which are specifically incorporated herein by reference in their entirety.
  • Antibodies of the disclosure also can be produced in a host cell transfectoma using, for example, a combination of recombinant DNA techniques and gene transfection methods as is well known in the art (e.g., Morrison, S. (1985) Science 229: 1202) .
  • DNA encoding partial or full-length light and heavy chains obtained by standard molecular biology techniques is inserted into one or more expression vectors such that the genes are operatively linked to transcriptional and translational regulatory sequences.
  • the term “operatively linked” is intended to mean that an antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the antibody gene.
  • regulatory sequence is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody genes.
  • promoters e.g., promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody genes.
  • enhancers e.g., polyadenylation signals
  • polyadenylation signals e.g., polyadenylation signals
  • Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV) , Simian Virus 40 (SV40) , adenovirus, e.g., the adenovirus major late promoter (AdMLP) and polyoma.
  • CMV cytomegalovirus
  • SV40 Simian Virus 40
  • AdMLP adenovirus major late promoter
  • nonviral regulatory sequences can be used, such as the ubiquitin promoter or ⁇ -globin promoter.
  • regulatory elements composed of sequences from different sources, such as the SR ⁇ promoter system, which contains sequences from the SV40 early promoter and the long terminal repeat of human T cell leukemia virus type 1 (Takebe et al., (1988) Mol. Cell. Biol. 8: 466-472) .
  • the expression vector and expression control sequences are chosen to be compatible with the expression host cell used.
  • the antibody light chain gene and the antibody heavy chain gene can be inserted into the same or separate expression vectors.
  • the variable regions are used to create full-length antibody genes of any antibody isotype by inserting them into expression vectors already encoding heavy chain constant and light chain constant regions of the desired isotype such that the V H segment is operatively linked to the C H segment (s) within the vector and the V L segment is operatively linked to the C L segment within the vector.
  • the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from a host cell.
  • the antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene.
  • the signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein) .
  • the recombinant expression vectors of the disclosure can carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes.
  • the selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216; 4,634,665 and 5,179,017) .
  • the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced.
  • Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells with methotrexate selection/amplification) and the neo gene (for G418 selection) .
  • DHFR dihydrofolate reductase
  • the expression vector (s) encoding the heavy and light chains is transfected into a host cell by standard techniques.
  • the various forms of the term “transfection” are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calcium-phosphate precipitation, DEAE-dextran transfection and the like.
  • Preferred mammalian host cells for expressing the recombinant antibodies of the disclosure include Chinese Hamster Ovary (CHO cells) (including dhfr-CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77: 4216-4220, used with a DHFR selectable marker, e.g., as described in R.J. Kaufman and P.A. Sharp (1982) J. Mol. Biol. 159: 601-621) , NSO myeloma cells, COS cells and SP2 cells.
  • Chinese Hamster Ovary CHO cells
  • dhfr-CHO cells described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77: 4216-4220
  • a DHFR selectable marker e.g., as described in R.J. Kaufman and P.A. Sharp (1982) J. Mol. Biol. 159: 601-621
  • another preferred expression system is the GS gene expression system disclosed in WO 87/04462, WO 89/01036 and EP 338, 841.
  • the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or, more preferably, secretion of the antibody into the culture medium in which the host cells are grown.
  • Antibodies can be recovered from the culture medium using standard protein purification methods.
  • the antibody or antigen binding portion thereof of the disclosure may be conjugated to a therapeutic agent to form an immunoconjugate such as an antibody-drug conjugate (ADC) .
  • Suitable therapeutic agents include cytotoxins, alkylating agents, DNA minor groove binders, DNA intercalators, DNA crosslinkers, histone deacetylase inhibitors, nuclear export inhibitors, proteasome inhibitors, topoisomerase I or II inhibitors, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics, and anti-mitotic agents.
  • the antibody and therapeutic agent preferably are conjugated via a linker cleavable such as a peptidyl, disulfide, or hydrazone linker.
  • the linker is a peptidyl linker such as Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys, Ala-Asn-Val, Val-Leu-Lys, Ala-Ala-Asn, Cit-Cit, Val-Lys, Lys, Cit, Ser, or Glu.
  • the ADCs can be prepared as described in U.S. Pat. Nos. 7,087,600; 6,989,452; and 7,129,261; PCT Publications WO 02/096910; WO 07/038,658; WO 07/051,081; WO 07/059,404; WO 08/083,312; and WO 08/103,693; U.S. Patent Publications 20060024317; 20060004081; and 20060247295; the disclosures of which are incorporated herein by reference.
  • the present disclosure features a bispecific molecule comprising the antibody or antigen binding portion thereof of the disclosure linked to at least one other functional molecule, e.g., another peptide or protein (e.g., another antibody or ligand for a receptor) to generate a bispecific molecule that binds to at least two different binding sites or target molecules.
  • another functional molecule e.g., another peptide or protein (e.g., another antibody or ligand for a receptor) to generate a bispecific molecule that binds to at least two different binding sites or target molecules.
  • the “bispecific molecule” includes molecules that have three or more specificities.
  • the bispecific molecules may be in many different formats and sizes. At one end of the size spectrum, a bispecific molecule retains the traditional antibody format, except that, instead of having two binding arms of identical specificity, it has two binding arms each having a different specificity. At the other extreme are bispecific molecules consisting of two single-chain antibody fragments (scFv′s) linked by a peptide chain, a so-called Bs (scFv) 2 construct. Intermediate-sized bispecific molecules include two different F (ab) fragments linked by a peptidyl linker. Bispecific molecules of these and other formats can be prepared by genetic engineering, somatic hybridization, or chemical methods.
  • the disclosure provides a chimeric antigen receptor comprising an anti-PD-L1 single chain variable fragment (scFv) comprising the heavy and light chain variable regions and/or CDRs of the disclosure.
  • scFv single chain variable fragment
  • the chimeric antigen receptor may comprise (a) an extracellular antigen recognition domain containing the anti-PD-L1 scFv, (b) a transmembrane domain, and (c) an intracellular signaling domain.
  • An oncolytic virus preferentially infects and kills cancer cells.
  • the antibody or antigen binding portion thereof of the disclosure may be used in conjunction with the oncolytic virus.
  • an oncolytic virus encoding the antibody or antigen binding portion thereof of the disclosure can be introduced into human body.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody or antigen binding portion thereof, the immunoconjugate, the bispecific molecule, the immune cell carrying the chimeric antigen receptor, the oncolytic virus, the nucleic acid molecule, the expression vector, and/or the host cell of the present disclosure formulated together with a pharmaceutically acceptable carrier.
  • the composition may optionally contain one or more additional pharmaceutically active ingredients, such as an anti-tumor agent, an anti-infective agent, or an agent for immunity enhancement.
  • the pharmaceutical composition of the disclosure may be administered in a combination therapy with, for example, an anti-tumor agent, an anti-infective agent, or an agent for immunity enhancement.
  • the pharmaceutical composition may comprise any number of excipients.
  • Excipients that can be used include carriers, surface active agents, thickening or emulsifying agents, solid binders, dispersion or suspension aids, solubilizers, colorants, flavoring agents, coatings, disintegrating agents, lubricants, sweeteners, preservatives, isotonic agents, and combinations thereof.
  • the selection and use of suitable excipients is taught in Gennaro, ed., Remington: The Science and Practice of Pharmacy, 20th Ed. (Lippincott Williams & Wilkins 2003) , the disclosure of which is incorporated herein by reference.
  • the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion) .
  • the active ingredient can be coated in a material to protect it from the action of acids and other natural conditions that may inactivate it.
  • parenteral administration means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.
  • an antibody of the disclosure can be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration, e.g., intranasally, orally, vaginally, rectally, sublingually or topically.
  • the pharmaceutical composition may be in the form of a sterile aqueous solution or dispersion.
  • the pharmaceutical composition may also be formulated in a microemulsion, liposome, or other ordered structure suitable for high drug concentration.
  • the amount of the active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated and the particular mode of administration and will generally be that amount of the composition which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 0.01%to about 99%of active ingredient in combination with a pharmaceutically acceptable carrier.
  • Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response) .
  • a single bolus can be administered, several divided doses can be administered over time or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.
  • parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • antibody can be administered as a sustained release formulation, in which case less frequent administration is required.
  • the dosage may range from about 0.0001 to 100 mg/kg body weight.
  • An exemplary treatment regime entails administration once per week.
  • Preferred dosage regimens for an anti-PD-L1 antibody of the disclosure include intravenous administration.
  • a “therapeutically effective dosage” of an anti-PD-L1 antibody of the disclosure preferably results in a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction.
  • a “therapeutically effective dosage” preferably inhibits tumor growth by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and still more preferably by at least about 80%relative to untreated subjects.
  • a therapeutically effective amount of a therapeutic antibody can decrease tumor size, or otherwise ameliorate symptoms in a subject, which is typically a human or can be another mammal.
  • the pharmaceutical composition can be a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
  • compositions can be administered via medical devices such as (1) needleless hypodermic injection devices (e.g., U.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; and 4,596,556) ; (2) micro-infusion pumps (U.S. Pat. No. 4,487,603) ; (3) transdermal devices (U.S. Pat. No. 4,486,194) ; (4) infusion apparatuses (U.S. Pat. Nos. 4,447,233 and 4,447,224) ; and (5) osmotic devices (U.S. Pat. Nos. 4,439,196 and 4,475,196) ; the disclosures of which are incorporated herein by reference.
  • medical devices such as (1) needleless hypodermic injection devices (e.g., U.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413;
  • the monoclonal antibodies of the disclosure can be formulated to ensure proper distribution in vivo.
  • they can be formulated in liposomes, which may additionally comprise targeting moieties to enhance selective transport to specific cells or organs. See, e.g. U.S. Pat. Nos. 4,522,811; 5,374,548; 5,416,016; and 5,399,331; V.V. Ranade (1989) J. Clin. Pharmacol. 29: 685; Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153: 1038; Bloeman et al., (1995) FEBS Lett.
  • the pharmaceutical composition of the disclosure may have numerous in vitro and in vivo utilities involving, for example, treatment and/or prevention of cancers and infectious diseases.
  • the pharmaceutical composition of the disclosure may be administered to human subjects, to inhibit tumor growth, or reduce or eliminate pathogens.
  • the disclosure provides methods for inhibiting growth of tumor cells in a subject comprising administering to the subject the pharmaceutical composition of the disclosure such that growth of the tumor is inhibited in the subject.
  • tumors that can be treated by antibodies of the disclosure include, but not limited to, melanoma, non-small cell lung cancer, renal cell carcinoma, Hodgkin lymphoma, bladder cancer, head and neck cancer, neuroendocrine tumor, mantle cell lymphoma, diffuse large B-cell lymphoma, and follicular lymphoma, original and/or metastatic. Additionally, refractory or recurrent malignancies may be inhibited using the pharmaceutical composition of the disclosure.
  • the disclosure provides a method for treating an infectious disease in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of the disclosure.
  • the infectious disease may be caused by viral, bacterial, fungal, or mycoplasma infection.
  • the infectious disease may be a chronic hepatitis B virus (HBV) , hepatitis C virus (HCV) , human immunodeficiency virus (HIV) , or simian immunodeficiency virus (SIV) infection.
  • the disclosure provides methods of combination therapy in which the pharmaceutical composition of the disclosure is co-administered with one or more additional antibodies or non-antibody agents that are effective in inhibiting tumor growth in a subject.
  • the disclosure provides a method for inhibiting tumor growth in a subject comprising administering to the subject the pharmaceutical composition of the disclosure with one or more additional antibodies, such as an anti-VISTA antibody, an anti-LAG-3 antibody, an anti-PD-1 antibody and/or an anti-CTLA-4 antibody.
  • the pharmaceutical composition of the disclosure may be used in combination with a chemotherapeutic agent, which is toxic to cells.
  • Other therapies that may be combined with anti-PD-L1 antibody includes, but not limited to, interleukin-2 (IL-2) administration, radiation, surgery, or hormone deprivation.
  • the subject is human.
  • the pharmaceutical composition of the disclosure may be used in combination with one or more other antibodies or non-antibody agents, to effectively reduce or eliminate pathogens in a subject, such as viruses, bacteria, fungi, or mycoplasmas.
  • the pharmaceutical composition of the disclosure may be used with an anti-infectious agent, including, but not limited to, an anti-virus agent, an anti-bacterial agent, an anti-fungal agent, and an anti-mycoplasma agent.
  • combination of therapeutic agents discussed herein can be administered concurrently as a single composition in a pharmaceutically acceptable carrier, or concurrently as separate compositions with each agent in a pharmaceutically acceptable carrier. In another embodiment, the combination of therapeutic agents can be administered sequentially.
  • sequential administration can be reversed or kept in the same order at each time point of administration, sequential administrations can be combined with concurrent administrations, or any combination thereof.
  • Example 1 Construction of HEK293A cell lines stably expressing human, monkey or mouse PD-L1
  • Cell lines stably overexpressing human, monkey or mouse PD-L1 were constructed using HEK293A cells (Cobioer, NJ, China) . Briefly, cDNA sequences respectively encoding human, monkey and mouse PD-L1 proteins (amino acid sequences set forth in SEQ ID NOs: 35, 36 and 37, respectively) were synthesized, and then subcloned into pLV-EGFP (2A) -Puro vectors. Lentiviruses were generated in HEK-293T cells (Cobioer, NJ, China) by cotransfection of pLV-EGFP (2A) -Puro-PD-L1, psPAX and pMD2.
  • HEK293A cells were infected with the lentiviruses to generate HEK293A cell lines stably expressing human, monkey and mouse PD-L1 respectively, namely HEK293A/human PD-L1 cells, HEK293A/monkey PD-L1 cells and HEK293A/mouse PD-L1 cells.
  • Transfected HEK293A cells were then cultured in medium (DMEM+10%FBS) containing 0.2 ⁇ g/ml puromycin (Cat#: A11138-03, Gibco) for 7 days.
  • the expressions of human PD-L1 and cynomolgus PD-L1 were confirmed by FACS using a commercially available anti-PD-L1 antibody (PE anti-human PD-L1 Antibody, Cat#: 393607, Biolegend, US) .
  • the expression of mouse PD-L1 was confirmed by FACS using a commercially available anti-mouse PD-L1 antibody (PE anti-mouse PD-L1 Antibody, Cat#: 124307, Biolegend, US) .
  • Example 2 Generation of hybridoma cell lines producing monoclonal mouse antibodies against human PD-L1
  • Murine anti-human PD-L1 monoclonal antibodies were generated using the conventional hybridoma fusion technology with some modifications as follows.
  • mice (Beijing Vital River Laboratory Animal Technology Co., Ltd, Beijing, China) were injected with recombinant human PD-L1 (ECD) -hFc (Cat#: 10084-H02H, Sino Biological, CN) and recombinant cynomolgus PD-L1 (ECD) -hFc (Cat#: 90251-C02H, Sino Biological, CN) following the scheme in Table 2 below.
  • the human PD-L1 (ECD) -hFc and cynomolgus PD-L1 (ECD) -hFc were emulsified by sonication with an equal volume of Complete Freund’s Adjuvant (Cat#: F5881-10*10ML, SIGMA, US) , Incomplete Freund’s Adjuvant (Cat#: F5506-6*10ML, SIGMA, US) , or PBS.
  • mice were used for hybridoma cell line generation.
  • Hybridoma cell lines were generated using the conventional hybridoma fusion technology with minor modifications as follows.
  • mice Four days after the final boost, mice were sacrificed, and spleens were collected and prepared as single cell suspensions in PBS.
  • the spleenocytes were washed for three times with DMEM medium (Cat#: SH30243.01B, Hyclone, US) .
  • Viable myeloma cells SP2/0 (CRL-1581, ATCC, US) at the log-phase were mixed with the murine spleenocytes at a ratio of 1: 4. The cells were then washed twice and then cell fusion was performed with PEG (Cat#: P7181, Sigma, US) .
  • the post-fusion cells were washed with DMEM medium for three times and suspended in cell growth medium (RPMI medium 1640 (Cat#: C22400500CP, Gibco) ) supplemented with 10%FBS and 1X HAT (Cat#: H0262, Sigma) .
  • the cell suspensions were plated onto 96 well cell culture plates, 200 ⁇ l per well, containing about 5 ⁇ 10 4 cells, and incubated in a 37°C humidified 5%CO 2 incubator for 7 days. Then, the growth medium was replaced by fresh one supplemented with 10%FBS and 1X HAT. Two to three days later, cell culture supernatants were collected for hybridoma cell screening by ELISA and FACS.
  • High-throughput ELISA binding assay was performed to screen for hybridoma clones producing monoclonal antibodies binding to human PD-L1, using human PD-L1 (ECD) -his (Cat#: 10084-H08H, Sino Biological, CN) .
  • the hybridoma clones that produced antibodies binding to human PD-L1 were further tested for their abilities to cross-react with cynomolgus PD-L1 using cynomolgus PD-L1 (ECD) -hFc (Cat#: 90251-C02H, Sino Biological, CN) .
  • the 249 hybridoma clones were further tested for their binding capabilities to human, cynomolgus and mouse PD-L1s expressed on HEK293A cells, using the HEK293A/human PD-L1 cells, HEK293A/monkey PD-L1 cells and HEK293A/mouse PD-L1 cells as prepared in Example 1.
  • the 88 hybridoma clones were subject to 2 rounds of subcloning. During the subcloning, multiple subclones (n>3) from each parent clone were selected and tested by ELISA and FACS assays as described above. The subclones selected through this process were defined as hybridoma cells producing monoclonal antibodies. Finally, 79 subclones (one subclone from each parent clone) having high binding capabilities to both human and monkey PD-L1 were obtained.
  • Monoclonal antibodies were then purified using a pre-equilibrated Protein-Aaffinity column (Cat#: 17040501, GE, US) and eluted with elution buffer (20 mM citric acid, pH3.0-pH3.5) . Then, antibodies were kept in PBS buffer (pH 7.0) , and their concentrations were determined using a NanoDrop instrument.
  • the isotype of each purified antibody was determined by using Rapid Isotyping Kit with Kappa and Lambda-Mouse (Cat#: 26179, Thermal, US) and Mouse Monoclonal Antibody Isotyping Reagents (Cat#: IS02-1KT, Sigma, US) , following the manufacturer’s manuals.
  • clones including 3C2 and 56E5, produced IgG1/kappa antibodies, while the remaining ones produced IgG2a/kappa or IgG2b/kappa antibodies.
  • the expression titers for clone 3C2 and 56E5 were 6.3 mg/L and 7.8 mg/L, respectively.
  • a cell-based binding assay was performed by FACS using the HEK293A cells stably overexpressing human, monkey and mouse PD-L1s respectively as generated in Example 1. Briefly, 10 5 HEK293A cells in 100 ⁇ l culture medium were seeded onto each well of the 96-well plates, to which added 50 ⁇ l serially diluted anti-PD-L1 antibodies. After incubated at 4°C for 1 hour, plates were washed 3 times with PBST.
  • APC coupled Goat Anti-Mouse IgG (Cat#: 405308, BioLegend, US) diluted 500x was added to the plates. After incubation at 4°C for 1 hour, the plates were washed with PBS for 3 times and then cell fluorescence was monitored using a FACS machine (BD) .
  • mice anti-PD-L1 monoclonal antibodies showed high binding capabilities to both human and monkey PD-L1s, but did not bind mouse PD-L1.
  • EC 50 values of the representative antibodies were summarized in Table 3 below.
  • a competition ELISA assay was performed. Briefly, 96-well plates were coated with 0.5 ⁇ g/ml Human PD-L1 (ECD) -His (Cat#: 10084-H08H, Sino Biological, CN) , 100 ⁇ l per well, at 4°C overnight. The wells were blocked with 200 ⁇ l blocking buffer (PBS containing 1%BSA, 1%goat serum and 0.05%Tween 20) for 2 hours at room temperature.
  • ECD Human PD-L1
  • His Cat#: 10084-H08H, Sino Biological, CN
  • Atezolizumab prepared using the amino acid sequences disclosed in WO2020226986 with human IgG1 (N297A) /kappa constant regions
  • Avelumab prepared using the amino acid sequences disclosed in WO2013079174A1 with human IgG1/kappa constant regions
  • Durvalumab prepared using the amino acid sequences disclosed in US20190276543A1 with human IgG1 (L234F, L235E, P331S) /kappa constant regions
  • anti-Hel antibody Cat#: LT12031, LifeTein, US
  • the plates were incubated for 1 hour at room temperature, washed for 3 times with PBST, added with 100 ⁇ l 1 ⁇ g/ml antibodies of the disclosure, and incubated for 1 hour at room temperature.
  • the ELISA plates were washed for 3 times with PBST, added with anti-mouse Fc-HRP (Cat#: A9309-1MC, Sigma, US) diluted at 1: 20000, and incubated for 1 hour at room temperature. Plates were washed by PBST for 3 times, and developed with freshly prepared Ultra-TMB (Cat#: TMB-S-003, Huzhou Yingchuang, CN) for 5 minutes at room temperature.
  • the absorbance was measured in a microplate reader (Thermo Multiscan FC) at 450 nm.
  • mice did not compete with atezolizumab, avelumab or durvalumab over epitope binding, indicating that they bound to different epitopes compared to the reference antibodies.
  • the antibodies from clone 56E5 competed with all three reference antibodies over epitope binding, indicating that the 56E5 antibodies bound to same or similar epitopes as the references did.
  • PD-1 is the receptor for PD-L1.
  • a cell-based blocking assay was performed by FACS to measure the antibodies’ blocking capabilities on PD-1-PD-L1 interaction, using the HEK293A cells stably overexpressing human PD-L1 as generated in Example 1. Briefly, 10 5 HEK293A/human PD-L1 cells in 100 ⁇ l culture medium were seeded onto each well of 96-well plates, to which added 50 ⁇ l serially diluted anti-PD-L1 antibodies. After incubated at 4°C for 1 hour, the plates were washed 3 times with PBST.
  • PD1-hFc proteins (Cat#: 10377-H02H, Sino Biological, CN) were added into the plates, 100 ⁇ l per well. After incubation at 4°C for 1 hour, the plates were washed with PBST for 3 times, and added with PE coupled Goat Anti-human IgG (Cat#: PAI-86078, Thermofisher, US) diluted 500x. After incubation at 4°C for 1 hour, the plates were washed with PBST for 3 times and then cell fluorescence was monitored using a FACS machine (BD) .
  • BD FACS machine
  • mouse anti-PDL1 antibodies on APC-mediated T cell activation was studied in a mixed lymphocyte reaction (MLR) assay.
  • MLR mixed lymphocyte reaction
  • PBMCs were collected from one healthy human donor’s blood sample by density gradient centrifugation, and then re-suspended in RPMI1640 medium.
  • PBMCs were cultured in a 37°C incubator for 2 hours, and cells adhered to container walls were collected as isolated monocytes.
  • the monocytes were cultured in RPMI1640 medium supplemented with 10%FBS, 100 ng/ml recombinant human GM-CSF (Cat#: 7954-GM, R&D, US) and 100 ng/ml recombinant human IL-4 (Cat#: 6507-IL, R&D, US) . Three days later, half of the medium was replaced with fresh one.
  • the culture medium was replaced by fresh medium containing 100 ng/ml recombinant human GM-CSF, 100 ng/ml recombinant human IL-4, 10 ng/ml rhTNF- ⁇ (Cat#: 210-TA-100, R&D, US) , 1000 U/ml rhIL-6 (Cat#: 7270-IL-025, R&D, US) , 1 ⁇ g/ml PGE2 (Cat#: 363-24-6, TOCRIS, US) and 10 ng/ml IL-1 ⁇ (Cat#: 210-LB-025, R&D, US) .
  • the cells were cultured for another 2 days.
  • PBMCs from another healthy human donor’s blood sample were collected by density gradient centrifugation and then re-suspended in RPMI1640 medium.
  • CD4 + T cells were isolated from the PBMCs using Invitrogen Dynabeads Untouched Human CD4 + T cells isolation kit (Cat#: 11346D, Thermal Fisher Scientific, US) , according to the manufacturer′s instructions.
  • the dendritic cells from the first donor and the CD4 + T cells from the second donor were seeded on 96 well U-bottom plates respectively at 2.5 ⁇ 10 4 cells/well and 5 ⁇ 10 4 cells/well, in a total of 150 ⁇ l culture medium per well.
  • the plates were added with 50 ⁇ l anti-PD-L1 antibodies (0.1-10 ⁇ g/ml) , or the anti-Hel control (Cat#: LT12031, LifeTein, US) , and further incubated for 72 hours.
  • IFN- ⁇ concentration was determined by an ELISA kit (Cat#: SIF50, R&D, US) following the manufacturer′s protocol. The assay was done in triplicate.
  • FIG. 1 (A) highest IFN- ⁇ levels were detected in wells treated with 3C2 and 56E5 antibodies. These antibodies increased IFN- ⁇ secretion by T cells compared to anti-Hel isotype control, in a dose dependent manner (FIG. 1 (B) ) .
  • Example 8 Expression and purification of chimeric anti-PD-L1 antibodies
  • the 3C2 and 56E5 antibodies were further studied.
  • the heavy/light chain variable region sequences of the two antibodies were cloned from hybridoma cells using the standard PCR method with a set of primers as describes in literatures (Juste et al., (2006) , Anal Biochem. 349 (1) : 159-61) , and sequenced. The sequences were summarized in Table 1 and Table 8.
  • Expression vectors were constructed by inserting the sequences encoding the variable region sequences plus human IgG1 (N297A) /kappa constant region sequences (amino acid sequences of heavy chain constant region and light chain constant region set forth in SEQ ID NOs: 33 and 34, respectively) into XhoI/BamHI restriction sites of pCDNA3.1 (Invitrogen, Carlsbad, US) .
  • the expression vectors were PEI transfected into HEK-293F cells (Cobioer, NJ, CN) .
  • HEK-293F cells were cultured in Free Style TM 293 Expression Medium (Cat#: 12338-018, Gibco) and transfected with the expression vectors using polyethyleneinimine (PEI) at a DNA: PEI ratio of 1: 3, 1.5 ⁇ g DNAs per millimeter of cell medium.
  • PEI polyethyleneinimine
  • Transfected HEK-293F cells were cultured in an incubator at 37°C under 5%CO 2 with shaking at 120 RPM. After 10-12 days, supernatants were harvested and monoclonal antibodies were purified as described in Example 3.
  • the chimeric anti-PD-L1 antibodies were further characterized for their abilities of binding to human PD-L1, monkey PD-L1 and mouse PD-L1. Briefly, ELISA plates were coated with 500 ng/ml human PD-L1 (ECD) -his (Cat#: 10084-H08H, Sino Biological, CN) , 100 ⁇ l per well, at 4°C overnight. The wells were blocked with 200 ⁇ l blocking buffer (PBS containing 1%BSA, 1%goat serum and 0.05%Tween 20) for 2 hours at room temperature, added and incubated with 100 ⁇ l serially diluted anti-PD-L1 antibodies (starting at 40 ⁇ g/ml) for 1 hour at room temperature.
  • PBS containing 1%BSA, 1%goat serum and 0.05%Tween 20
  • the plates were washed for 3 times with PBST (PBS+0.05%Tween 20) , added with Goat-anti-human IgG-HRP (Cat#: 31410, Thermal, US) diluted 5000X, and incubated for 1 hour at room temperature. Plates were developed with freshly prepared Ultra-TMB (Cat#: 555214, BD, US) for 5 minutes at room temperature. Absorbance was read on a SpectraMax R i3X reader (Molecular Devies, US) at 450 nm.
  • Species-cross-reactivities of the anti-PD-L1 mAbs to monkey or mouse PD-L1s were further assessed by direct ELISA. Briefly, 96-well ELISA plates were coated with 500 ng/ml monkey PD-L1 (ECD) -his (Cat#: 90251-C08H, Sino Biological, CN) or mouse PD-L1 (ECD) -his (Cat#: 50010-M08H, Sino Biological, CN) , 100 ⁇ l per well, then added and incubated with 100 ⁇ l serially diluted anti-PD-L1 antibodies (starting at 40 ⁇ g/ml) .
  • the EC 50 values of the representative antibodies in the binding capability tests were summarized in Table 5. The data showed that all the chimeric antibodies had high binding capabilities to human and monkey PD-L1s, but did not bind mouse PD-L1. The binding capabilities of the chimeric 3C2 and 56E5 antibodies were comparable to those of their parent antibodies and higher than that of atezolizumab.
  • the antibodies were also tested for their binding capabilities to HEK293A/human PD-L1 cells, HEK293A/monkey PD-L1 cells and HEK293A/mouse PD-L1 cells as generated in Example 1, following the protocol of Example 4. The test results were shown in FIG. 2.
  • the chimeric antibodies had high binding capabilities to both human PD-L1 (FIG. 2 (A) ) and monkey PD-L1 (FIG. 2 (B) ) , but did not bind to mouse PD-L1 (FIG. 2 (C) ) .
  • Example 10 Chimeric anti-PD-L1 monoclonal antibodies inhibited PD-L1-PD-1 interaction
  • the chimeric anti-PD-L1 antibodies were further characterized for their blocking abilities on PD-1-PD-L1 interaction, using the HEK293A/human PD-L1 cells as generated in Example 1, following the protocol of Example 6. The test results were shown in FIG. 3.
  • the chimeric antibodies evidently blocked PD1-PD-L1 interaction, with chimeric 56E5 showing the best blocking effect, which was better than that of atezolizumab.
  • chimeric antibodies were further tested for their abilities to stimulate T cell response in a T cell function assay according to the protocol of Example 7.
  • IFN- ⁇ levels were determined using a commercially available kit (Cat#: STA00C, R&D, US) , following the manufacturer′s instruction.
  • the chimeric 56E5 antibody was more potent in T cell activation than atezolizumab at a low dose, e.g., 0.01 ⁇ g/mL.
  • the chimeric 3C2 antibody’s capability in T cell activation was higher than atezolizumab at all test doses.
  • 3C2 and 56E5 were humanized and further investigated. Humanization of the murine antibodies was conducted using the well-established CDR-grafting method (U.S. Pat. No. 5,225,539, incorporated herein by reference in its entirety) as described in detail below.
  • acceptor frameworks for humanization of murine antibodies 3C2 and 56E5 the light and heavy chain variable region sequences of 3C2 and 56E5 were blasted against the human immunoglobulin gene database in NCBI website (http: //www. ncbi. nlm. nih. gov/igblast/) .
  • the human germline IGVH and IGVK with the highest homology to 3C2 and 56E5 were selected as the acceptors for humanization.
  • the human heavy chain acceptor selected was IGHV1-46*01
  • the human light chain acceptor selected was IGKV1-33*01.
  • the human heavy chain acceptor selected was IGHV4-31*02
  • the human light chain acceptor selected was IGKV4-1*01.
  • the three dimensional structures were simulated for variable domains of 3C2 and 56E5, in order to identify key framework residues that might be playing important roles in supporting CDR loop structures, thus designing back mutations in humanized antibodies.
  • sequences encoding the heavy chain variable region plus human IgG1 constant region with N297A mutation and the sequences encoding the light chain variable regions plus human kappa constant region were chemically synthesized and then subcloned into GS expression vectors (Invitrogen, USA) using the EcoR I/Xho I and Cla I/Hind III restriction sites respectively. All expression constructs were confirmed by DNA sequencing.
  • the EXPiCHO expression systems (Invitrogen, USA) were transfected with heavy chain and light chain expressing vectors and transiently expressed 25 humanized anti-PD-L1 antibodies, following the protocol described in Example 8. The humanized antibodies were purified as described in Example 3.
  • the humanized anti-PD-L1 antibodies were characterized for their binding abilities to HEK293A/human PD-L1 cells, HEK293A/monkey PD-L1 cells and HEK293A/mouse PD-L1 cells, following the protocols described in Example 4. The results were shown in FIG. 5 and FIG. 6.
  • the humanized anti-PDL1 antibodies had high binding abilities to both human PD-L1 and monkey PD-L1, which were comparable to those of their respective chimeric antibodies.
  • the humanized 3C2 and 56E5 antibodies had comparable or higher binding affinities to human PD-L1 as compared to atezolizumab.
  • IFN- ⁇ levels were determined using a commercially available kit (Cat#: STA00C, R&D, US) following the manufacturer′s instructions.
  • a competition SPR assay was performed. Briefly, 1 ⁇ g/ml human PD-L1 (ECD) -his protein (Cat#: 10084-H08H, Sino Biological, CN) was coupled to CM5 biosensor chips (Cat#: BR-1005-30, GE Life Sciences, US) , and un-reacted groups were blocked with 1 M ethanolamine.
  • ECD human PD-L1
  • CM5 biosensor chips Cat#: BR-1005-30, GE Life Sciences, US
  • 5 ⁇ g/ml 56E5VH5VL4 or 3C2VH4VL4 antibodies were injected into the SPR running buffer (HBS-EP buffer, pH7.4, Cat#: BR-1006-69, GE Life Sciences, US) at 30 ⁇ L/minute, followed by a second anti-PDL1 antibody (56E5VH5VL4, 3C2VH4VL4, atezolizumab, avelumab or durvalumab) , 5 ⁇ g/ml, at 30 ⁇ L/minute.
  • the binding affinities were calculated with the RUs of blank controls subtracted. The data were then fitted using a 1: 1 interaction model.
  • 56E5VH5VL4 and atezolizumab did not bind the PD-L1 molecules at the same time, suggesting they may bind to the same or similar epitopes.
  • 56E5VH5VL4 competed with avelumab and furvalumab over epitope binding. While 56E5VH5VL4 and 3C2VH4VL4 simultaneously bound to the PD-L1 molecules, meaning they bound to different epitopes.
  • the antibody 3C2VH4VL4 bound to the PD-L1 molecules simultaneously with 56E5VH5VL4, atezolizumab, avelumab or furvalumab, suggesting that 3C2VH4VL4 may bind to a novel epitope different from those bound by the other antibodies.
  • mice were subcutaneously injected with 1 ⁇ 10 6 MC38 cells at one flank, and randomly allocated into 7 groups, 8 mice per group.
  • Tumor sizes and mice body weights were monitored over time.
  • the tumor size was determined by measuring by a caliper the length (the longest diameter) and width (the diameter perpendicular to the length) of a tumor and calculating the tumor volume as 0.5 x D x d 2 .
  • the test was terminated before the tumor sizes in the control group reached 3.5 cm 3 .
  • One-way ANOVA was used to identify tumor size differences among groups.

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Abstract

L'invention concerne un anticorps monoclonal isolé, ou une partie de liaison à l'antigène de celui-ci, qui se lie de manière spécifique à PD-L1 humain. L'invention concerne également une molécule d'acide nucléique codant pour l'anticorps ou une partie de liaison à l'antigène de celui-ci, un vecteur d'expression, une cellule hôte et une méthode pour exprimer l'anticorps ou une partie de liaison à l'antigène de celui-ci. La présente invention concerne en outre un immunoconjugué, une molécule bispécifique, un récepteur antigénique chimérique, un virus oncolytique et une composition pharmaceutique comprenant l'anticorps ou une partie de liaison à l'antigène de celui-ci, ainsi qu'une méthode de traitement faisant appel à l'anticorps ou à une partie de liaison à l'antigène de celui-ci.
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